Water-soluble thymine compounds



synthesized by several methods.

Patented June 26, 1951 WATER-SOLUBLE THYMINE COMPOUNDS Karl Dittmcr, Irving Goodman, and Donald Visser, Boulder, 0010., assignors to The Chemical Foundation, Inc.,.New York, N. Y., a corporation of New York No Drawing. Application April 8, 1947, Serial No. 740,260

4 Claims. (01. 260-2115) This invention relates to biologically active water soluble thymine compounds, more particularly to the synthesis of hexoso and pentcso thymines and their use as specific food factors.

While it is known that thymine is necessary for the growth of certain bacteria, namely, the Lactobacilli, the fact that it is poorly water soluble naturally imposes marked limitations on its biological usefulness.

As is known, thymine was discovered by Kossel and Newman in 1893 and was successfully synthesized in 1901. This compound. has since been The compound has recently assumed marked importance due to the report by Spies (Southern Medical Journal 39, 269-270, 1946) of its beneficial effect in the treatment of pernicious anemia. This investigator reported that the dosage required is several thousand times that of folic acid when the latter is used for treating the disease. Thus, thymine can be substituted for folic acid in the treatment of the disease but the dose required is inordinately high. Similarly, it has been reported that thymine (Keatha et al., Jour. Biol. Chem. 158, 145- 156, 1945) may be substituted for folic acid (L. casez factor) in influencing the growth of S. lactis R. In the same report there was shown to be a further substitution of thymine for folic acid in the support of the growth of L. casei organisms.

In the above reports thymine (2,6-dioxy-5- methyl pyrimidine; 5 methyl uracil) was used but it is to be noted that, as such, the compound is poorly water soluble rendering it difiicult in its assimilation by the organism. Thymine is a crystalline powder, made up of short white crystals, melting with decomposition at 320 0., is very slightly soluble in ether and difficultly soluble in water and alcohol. The potassium, sodium, silver, mercury and lead salts of the compound are likewise sparingly water soluble. Thus, to date, the solubility of thymine has been a problem with reference to its use in biological systems.

It has now been found that highly water soluble thymine compounds may be produced which present marked biological activity.

In the course of intensive investigation in this field it has been ascertained that thymine may be coupled with certain sugars to produce a readily water soluble compound of enhanced biological activity. It has been determined specifically, for example, that 3-glucosido thymine exerts a high biologic activity for L. casez' whereas its molecular equivalent of thymine exerts but a low degree of activity. Furthermore, 3-glucosido thymine exerts a beneficial eifect on sulfaguanidine in- 2 duced macrocytic anemias in rats, whereas thymine alone is less effective.

Schmidt, Nickels and Johnson, although not seeking glucosido thymine, endeavored to synthesize 3-methyl thymine (J. A. C. S. 52, 4511, 1930) from brom-aceto-glucose and 2,6 diethoxy thymine which resulted in a product having a melting point of 316 C. The yield was too small for analysis. Generally the synthesis carried out by Schmidt, Nickels and Johnson consisted of heating a mixture of 2 gms. of 2,6-dimethoxy-5 methylpyrimidine and 2 gms. of bromoacetoglucose. The resulting product was extracted with 5 ml. of water and the small portion which did not dissolve was presumed to be 3-methy1 thymine. Using the principle suggested by the above investigators the experiment was repeated with bromo aceto chloroaceto glucose, pyrimidine and 2,6 diethoxy-5-methylpyrimidine. However, our attempt to reproduce these results with larger quantities of material yielded a compound having a melting point of 326 C. which was identified as thymine.

The procedure we therefore had to employ for the preparation of 3 glucosido thymine was based in part on a principle developed by Hilbert and Johnson (J. A. C. S. 52, 4489, 1930) in their preparation of glucosido uracil. Their investigation demonstrated the possibility of applying a method of alkylation which enabled one to control a substitution in the 3-position of the pyrimidine ring. This involved a reaction between 2,6 dialkoxypyrimidine and an alkyl halide which interact in accordance with the equation:

N=CIJOC H5 CzHaO EIJH E (121150 CH 00 (3H 3 -CH H CHaN-CH-I-GzHsI They reported an analogous reaction between 2,6 dimethoxy pyrimidine and acetobromoglucose yielding 2-oxy-6-methoxy-3 tetra-acetylglucosido pyrimidine. The resultant 2-oxy-3-methyl-6- methoxy pyrimidine (liberated by the presence of methyl bromide reacting with 2,6 dimethoxy pyrimidine) and the acetylated glucosido (2-oxy- 6-methoxy-3-tetra acetylglucosido-pyrimidine) are separated and the latter de-ethylated and deacetylated on treatment with alcoholic hydrochloric acid yielding B-glucosido uracil.

Utilizing this principle, but substituting thymine for uracil, modifications involving these previous methods were employed. Because of the tendency of diethoxy thymine to react with ethyl 2,558,480 3 4 bromide, which is split off from the quaternary under vacuum and in the presence of pyridine to complex of diethoxy thymine and brom-aceto produce 3-aceto-aldoso-6-ethoxy thymine, then glucose, a vacuum of 2-3 mm. Hg was used. hydrolyzing such product under vacuum with Furthermore, to neutralize traces of acid present, aloohouc hydrochloric acid to de-acetylate and few drops of py W added 170 the reaction 5 de-ethylate the intermediate product and recovmixture. The reaction was carried out at 50 C. ering a water soluble 3-aldosido thymine.

for days at 2-3 H u ing the first 3 2. A method of producing a water soluble thydays of reaction considerable amounts of C2H5Br mine compound which comprises, reacting 2,6-di- Were iven off as evidenced by bu in the ethoxy-thymine with acetobromoglucose under reaction mixture and by analysis of the liquor 10 vacuum and in the presence of pyridine to proaccumulated in the solid CO2 acetone trap. The duce 3-acetoglucose-6-ethoxy thymine, then hyaddition of an equal volume of ether and cooling drolyzing such product under v u it alcm in a refrigerator for two months failed to proholic hydrochloric acid to de-acetylate and duce any crystals of the desired product which de-ethylate the intermediate product and recovper contra occurred in the case of diethoxy uracil ering a water soluble 3-glucosido thymine. and bromoaceto-glucose. Other standard meth- 3, A method of producing a water soluble thy- Ods for Obtaining ystals likewise failed. minecompound which comprises, reacting 2,6-di- If after hy olys s of t e ction mixture ith ethoxy thymine with acetobromo galactose under H01 in absolute methanol both CI-laOH and vacuum and in the presence of pyridine to CH3COOI-I are removed under reduced pressure produce 3-acetogalactose-6-ethoxy thymine, then and absolute ethanol and ether introduced pure hydrolyzing such product under vacuum with white crystals formed, which on analysis, were alcoholic hydrochloric acid to de-acetylate and revealed to be 3 glucosido thymine. Furtherde-ethylate the intermediate product and recov more, 3-glucosido thymine can be recrystallized ering water soluble 3-ga1actosido thymine. from 90% ethanol. The reaction can be. ex- 4. A method of producing a water soluble thypressed as follows: mine compound which comprises, reacting 2,6-di- AC AC 'Ac H|5C20C=N t n t 0 CH8 Ito-0255+ Br- J-|- -o H N V (I) H H:

(Diethoxy thymine) (Brom acetoglucose) mono-0:1 AC AC /AC HO-C=N o OHS-c 0:0 5 H t o deactylation ens-c E= OH H on on (1]) (l) (I; (I; deethylation H- N-- -o H- -NCC-O- -o I I i (alcoholic H01) I I E it o n E E1 on 11 to LC 7 (3 glucosido thymine) (3 acetoglucoso ethoxy thymine) The melting point of the final product is 263- ethoxy thymine with acetobromo arabinose under 266 C. (uncorrected), the solubility corresponds vacuum and in the presence of pyridine to proroughly to that of 3 glucosido uracil; N (found) duce 3-acetoarabinose-6-ethoxy thymine, then 9.53%; N (theory) =9.'72%. hydrolyzing such product under vacuum with Corresponding 3 saccharido thymines were prealcoholic hydrochloric acid to de-acetylate and pared with d-galactose, and d-arabinose by simi- 5o de-ethylate the intermediate product and recovlar methods where substitution in the 3-position ering water soluble 3-arabinosido thymine.

corresponded to process involving acetoglucose. KARL DITTMER. The biologic activity of these latter complexes of IRVING GOODMAN. thymine are as yet undetermined. DONALD VISSER.

It will be appreciated that the availability of a water soluble thymine compound presents REFERENCES CITED many advantages The product may be dissolved The following references are of record in the in physiological salt solutions and thus be made file of this patent;

available for parenteral use. Similarly, the product may be prepared in any suitable form for oral UNITED STALES PATENTS administration by incorporating it in pills and Number Name Date the hke, either alone or in association with other 2,134,463 Taylor et al Oct. 25, 1938 valuable therapeutic agents. As pointed out 2,336,890 Riegel et al Dec. 14, 1943 previously, the new water soluble thymine compounds are particularly useful as adjuvants in 55 OTHER REFERENCES culture media where bacteria specifically require Hilbert 811 841.1 Journ- Amer- Chemsuch a factor for their growth. pp- 4489 o 4 (NOV- 1930)- We claim: Schmidt-Nickels et al.: Journ. Amer. Chem.

1. A method of producing a water soluble thy- DD- 11 t0 1 19 0) mme compound which comprises reacting 2,6-dio y n me a har y Lippincctt ethoxy-thymine with a halogenated aceto-aldose 0 p g 

1. A METHOD OF PRODUCING A WATER SOLUBLE THYMINE COMPOUND WHICH COMPRISES REACTING 2,6-DIETHOXY-THYMINE WITH A HALOGENATED ACETO-ALDOSE UNDER VACUUM AND IN THE PRESENCE OF PYRIDINE TO PRODUCE 3-ACETO-ALDOSO-6-ETHOXY THYMINE, THEN HYDROLYZING SUCH PRODUCT UNDER VACUUM WITH ALCOHOLIC HYDROCHLORIC ACID TO DE-ACETYLATE AND DE-ETHYLATE THE INTERMEDIATE PRODUCT AND RECOVERING A WATER SOLUBLE 3-ALDOSIDO THYMINE. 